Cell Biochemistry and Biophysics

, Volume 67, Issue 3, pp 1249–1259 | Cite as

In Vitro Experimental Study for the Determination of Cellular Axial Strain Threshold and Preferential Axial Strain from Cell Orientation Behavior in a Non-uniform Deformation Field

  • Yasuyuki Morita
  • Sachi Watanabe
  • Yang Ju
  • Shuhei Yamamoto
Original Paper

Abstract

Cells within connective tissues are routinely subjected to a wide range of non-uniform mechanical loads that regulate many cell behaviors. In the present study, the relationship between cell orientation angle and strain value of the membrane was comprehensively investigated using an inhomogeneous strain field. Additionally, the cellular axial strain threshold, which corresponds to the launching of cell reorientation response, was elucidated. Human bone marrow mesenchymal stem cells were used for these experiments. In this study, an inhomogeneous strain distribution was easily created by removing one side holes of an elastic chamber in a commonly used uniaxial stretching device. The strains of 2D stretched membranes were quantified on a position-by-position basis using the digital image correlation method. The normal strain in the direction of stretch was changed continuously from 2.0 to 15.0 %. A 3D histogram of the cell frequency, which was correlated with the cell orientation angle and normal strain of the membrane, made it possible to determine the axial strain threshold accurately. The value of the axial strain threshold was 4.4 ± 0.3 %, which was reasonable compared with previous studies based on cyclic uniaxial stretch stimulation (homogeneous strain field). Additionally, preferential axial strain of cells, which was a cell property firstly introduced, was also achieved and the value was −2.0 ± 0.1 %. This study is novel in three respects: (i) it precisely and easily determined the axial strain threshold of cells; (ii) it is the first to suggest preferential axial strain of cells; and (iii) it methodically investigated cell behavior in an inhomogeneous strain field.

Keywords

2D strain 3D histogram Axial strain threshold Cell orientation Digital image correlation (DIC) Inhomogeneous deformation Mechanical stretch Mesenchymal stem cell (MSC) Strain field 

References

  1. 1.
    Ingber, D. E. (1997). Tensegrity: The architectural basis of cellular mechanotransduction. Annual Review of Physiology, 59, 575–599.PubMedCrossRefGoogle Scholar
  2. 2.
    Giannone, G., & Sheetz, M. P. (2006). Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways. Trends in Cell Biology, 16(4), 213–223.PubMedCrossRefGoogle Scholar
  3. 3.
    Tipton, C. M., Matthes, R. D., Maynard, J. A., & Carey, R. A. (1975). Influence of physical-activity on ligaments and tendons. Medicine & Science in Sports & Exercise, 7(3), 165–175.CrossRefGoogle Scholar
  4. 4.
    Woo, S. L. Y., Gomez, M. A., Woo, Y. K., & Akeson, W. H. (1982). Mechanical-properties of tendons and ligaments. II. The relationships of immobilization and exercise on tissue remodeling. Biorheology, 19(3), 397–408.PubMedGoogle Scholar
  5. 5.
    Wang, H. C., Ip, W., Boissy, R., & Grood, E. S. (1995). Cell orientation response to cyclically deformed substrates: Experimental validation of a cell model. Journal of Biomechanics, 28(12), 1543–1552.PubMedCrossRefGoogle Scholar
  6. 6.
    Neidlinger-Wilke, C., Grood, E. S., Wang, J. H. C., Brand, R. A., & Claes, L. (2001). Cell alignment is induced by cyclic changes in cell length: Studies of cells grown in cyclically stretched substrates. Journal of Orthopaedic Research, 19(2), 286–293.PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang, L., Kahn, C. J. F., Chen, H. Q., Tran, N., & Wang, X. (2008). Effect of uniaxial stretching on rat bone mesenchymal stem cell: Orientation and expressions of collagen types I and III and tenascin-C. Cell Biology International, 32(3), 344–352.PubMedCrossRefGoogle Scholar
  8. 8.
    Morita, Y., Watanabe, S., & Ju, Y. Determination of optimal cyclic uniaxial stretches for stem cell-to-tenocyte differentiation under a wide range of mechanical stretch conditions by evaluating gene expression and protein synthesis levels. Acta of Bioengineering and Biomechanics (accepted).Google Scholar
  9. 9.
    Arold, S. P., Wong, J. Y., & Suki, B. (2007). Design of a new stretching apparatus and the effects of cyclic strain and substratum on mouse lung epithelial-12 cells. Annals of Biomedical Engineering, 35(7), 1156–1164.PubMedCrossRefGoogle Scholar
  10. 10.
    Raeber, G. P., Lutolf, M. P., & Hubbell, J. A. (2008). Part II: Fibroblasts preferentially migrate in the direction of principal strain. Biomechanics and Modeling in Mechanobiology, 7(3), 215–225.PubMedCrossRefGoogle Scholar
  11. 11.
    Breen, E. C., Fu, Z. X., & Normand, H. (1999). Calcyclin gene expression is increased by mechanical strain in fibroblasts and lung. The American Journal of Respiratory Cell and Molecular Biology, 21(6), 746–752.CrossRefGoogle Scholar
  12. 12.
    Yang, G. G., Crawford, R. C., & Wang, J. H. C. (2004). Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions. Journal of Biomechanics, 37(10), 1543–1550.PubMedCrossRefGoogle Scholar
  13. 13.
    Koike, M., Shimokawa, H., Kanno, Z., Ohya, K., & Soma, K. (2005). Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2. Journal of Bone and Mineral Metabolism, 23(3), 219–225.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen, Y. J., Huang, C. H., Lee, I. C., Lee, Y. T., Chen, M. H., & Young, T. H. (2008). Effects of cyclic mechanical stretching on the mRNA expression of tendon/ligament-related and osteoblast-specific genes in human mesenchymal stem cells. Connective Tissue Research, 49(1), 7–14.PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang, L., Tran, N., Chen, H. Q., Kahn, C. J. F., Marchal, S., Groubatch, F., et al. (2008). Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching. Cell and Tissue Research, 332(1), 101–109.PubMedCrossRefGoogle Scholar
  16. 16.
    Xu, B., Song, G., & Ju, Y. (2011). Effect of focal adhesion kinase on the regulation of realignment and tenogenic differentiation of human mesenchymal stem cells by mechanical stretch. Connective Tissue Research, 52(5), 373–379.PubMedCrossRefGoogle Scholar
  17. 17.
    Xu, B., Song, G., Ju, Y., Li, X., Song, Y., & Watanabe, S. (2012). RhoA/ROCK, cytoskeletal dynamics and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells. Journal of Cellular Physiology, 227(6), 2722–2729.PubMedCrossRefGoogle Scholar
  18. 18.
    Katsumi, A., Naoe, T., Matsushita, T., Kaibuchi, K., & Schwartz, M. A. (2005). Integrin activation and matrix binding mediate cellular responses to mechanical stretch. Journal of Biological Chemistry, 280(17), 16546–16549.PubMedCrossRefGoogle Scholar
  19. 19.
    Buck, R. C. (1980). Reorientation response of cells to repeated stretch and recoil of the substratum. Experimental Cell Research, 127(2), 470–474.PubMedCrossRefGoogle Scholar
  20. 20.
    Buckley, M. J., Banes, A. J., Levin, L. G., Sumpio, B. E., Sato, M., Jordan, R., et al. (1988). Osteoblasts increase their rate of division and align in response to cyclic, mechanical tension in vitro. Bone Mineral, 4(3), 225–236.PubMedGoogle Scholar
  21. 21.
    Dartsch, P. C., & Hammerle, H. (1986). Orientation response of arterial smooth muscle cells to mechanical stimulation. European Journal of Cell Biology, 41(2), 339–346.PubMedGoogle Scholar
  22. 22.
    Gijsen, F. J. H., Wentzel, J. J., Thury, A., Lamers, B., Schuurblers, J. C. H., Serruys, P. W., et al. (2007). A new imaging technique to study 3-D plaque and shear stress distribution in human coronar arter bifurcations in vivo. Journal of Biomechanics, 40(11), 2349–2357.PubMedCrossRefGoogle Scholar
  23. 23.
    Banes, A. J., Link, G. W., Gilbert, J. W., Tay, R. T. S., & Monbureau, O. (1990). Culturing cells in a mechanically active environment. American Biotechnology Laboratory, 8(7), 12–22.PubMedGoogle Scholar
  24. 24.
    Iba, T., & Sumpio, B. E. (1992). Tissue plasminogen activator expression in endothelial cells exposed to cyclic strain in vitro. Cell Transplantation, 1(1), 43–50.PubMedGoogle Scholar
  25. 25.
    Wilson, E., Mai, Q., Sudhir, K., Weiss, R. H., & Ives, H. E. (1993). Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. Journal of Cell Biology, 123(3), 741–747.PubMedCrossRefGoogle Scholar
  26. 26.
    Gilbert, J. A., Weinhold, P. S., Banes, A. J., Link, G. W., & Jones, G. L. (1994). Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. Journal of Biomechanics, 27(9), 1169–1177.PubMedCrossRefGoogle Scholar
  27. 27.
    Awolesi, M. A., Sessa, W. C., & Sumpio, B. E. (1995). Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. The Journal of Clinical Investigation, 96(3), 1449–1454.PubMedCrossRefGoogle Scholar
  28. 28.
    Balestrini, J. L., Skorinko, J. K., Hera, A., Gaudette, G. R., & Billiar, K. L. (2010). Applying controlled non-uniform deformation for in vitro studies of cell mechanobiology. Biomechanics and Modeling in Mechanobiology, 9(3), 329–344.PubMedCrossRefGoogle Scholar
  29. 29.
    Yung, Y. C., Vandenburgh, H., & Mooney, D. J. (2009). Cellular strain assessment tool (CSAT): Precision-controlled cyclic uniaxial tensile loading. Journal of Biomechanics, 42(2), 178–182.PubMedCrossRefGoogle Scholar
  30. 30.
    Richardson, W. J., Metz, R. P., Moreno, M. R., Wilson, E., & Moore, J. E. (2011). A device to study the effects of stretch gradients on cell behavior. The Journal of Biomechanical Engineering ASME, 133(10), 101008.CrossRefGoogle Scholar
  31. 31.
    Aroush, D. R. B., Barlam, D., & Wngner, H. D. (2012). Generating an inhomogeneous stress field as a technique to study cell mechanoresponse. Applied Physics Letters, 100(13), 133703.CrossRefGoogle Scholar
  32. 32.
    Kelly, D. J., Azeloglu, E. U., Kochupura, P. V., Sharma, G. S., & Gaudette, G. R. (2007). Accuracy and reproducibility of a subpixel extended phase correlation method to determine micron level displacements in the heart. Medical Engineering & Physics, 29(1), 154–162.CrossRefGoogle Scholar
  33. 33.
    Bruck, H. A., McNeill, S. R., Sutton, M. A., & Peters, W. H. (1989). Digital image correlation using Newton-Raphson method of partial differential correction. Experimental Mechanics, 29(3), 261–267.CrossRefGoogle Scholar
  34. 34.
    Sutton, M. A. (2008). Digital image correlation for shape and deformation measurements. In W. N. Sharpe (Ed.), Springer handbook of experimental solid mechanics (pp. 565–600). New York: Springer.CrossRefGoogle Scholar
  35. 35.
    Kato, T., Ishiguro, N., Iwata, H., Kojima, T., Ito, T., & Naruse, K. (1998). Up-regulation of COX2 expression by uni-axial cyclic stretch in human lung fibroblast cells. Biochemical and Biophysical Research Communications, 244(3), 615–619.PubMedCrossRefGoogle Scholar
  36. 36.
    Inoh, H., Ishiguro, N., Sawazaki, S. I., Amma, H., Miyazu, M., Iwata, H., et al. (2002). Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor B in human fibroblast cells. FASEB Journal, 16(1), 405.PubMedGoogle Scholar
  37. 37.
    Amma, H., Naruse, K., Ishiguro, N., & Sokabe, M. (2005). Involvement of reactive oxygen species in cyclic stretch-induced NF-kappa B activation in human fibroblast cells. British Journal of Pharmacology, 145(3), 364–373.PubMedCrossRefGoogle Scholar
  38. 38.
    Wang, J. G., Miyazu, M., Xiang, P., Li, S. N., Sokabe, M., & Naruse, K. (2005). Stretch-induced cell proliferation is mediated by FAK-MAPK pathway. Life Sciences, 76(24), 2817–2825.PubMedCrossRefGoogle Scholar
  39. 39.
    Takeda, H., Komori, K., Nishikimi, N., Nimura, Y., Sokabe, M., & Naruse, K. (2006). Bi-phasic activation of eNOS in response to uni-axial cyclic stretch is mediated by differential mechanisms in BAECs. Life Sciences, 79(3), 233–239.PubMedCrossRefGoogle Scholar
  40. 40.
    Gilbert, J. A., Banes, A. J., Link, G. W., & Jones, G. L. (1990). Video analysis of membrane strain: An application in cell stretching. Experimental Techniques, 14(5), 43–45.CrossRefGoogle Scholar
  41. 41.
    Wang, J. H. C. (2006). Mechanobiology of tendon. Journal of Biomechanics, 39(9), 1563–1582.PubMedCrossRefGoogle Scholar
  42. 42.
    Costa, K. D., Hucker, W. J., & Yin, F. C. P. (2002). Buckling of actin stress fibers: A new wrinkle in the cytoskeletal tapestry. Cell Motility and the Cytoskeleton, 52(4), 266–274.PubMedCrossRefGoogle Scholar
  43. 43.
    Naruse, K., Yamada, T., & Sokabe, M. (1998). Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. American Journal of Physiology - Heart and Circulatory Physiology, 274(5), H1532–H1538.Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Yasuyuki Morita
    • 1
  • Sachi Watanabe
    • 1
  • Yang Ju
    • 1
  • Shuhei Yamamoto
    • 1
  1. 1.Department of Mechanical Science & Engineering, Graduate School of EngineeringNagoya UniversityNagoyaJapan

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